Erythropoietin, known to stimulate erythroid progenitor cell survival, proliferation, and differentiation, has been shown to be neuroprotective against brain ischemia in animal models. Both erythropoietin and its receptor are expressed in the brain and are up-regulated by hypoxia. Brain erythropoietin signaling can stimulate neural cell survival and prevent neuron apoptosis. Neurons from erythropoietin receptor null mice exhibit marked increased sensitivity to hypoxia. Erythropoietin has been shown to stimulate nitric oxide production in endothelial cells, particularly at low oxygen tension. We found that the neural protective effect of erythropoietin is regulated by nitric oxide. Hypoxia increased nitric oxide production and erythropoietin receptor expression in neural cultures, and inhibition of nitric oxide synthase activity reduced the proportion of erythropoietin receptor-expressing neurons induced at low oxygen tension. Conversely, a nitric oxide donor induced erythropoietin receptor expression in neural cultures and increased erythropoietin receptor promoter activity in reporter gene assays, indicating regulation at the transcription level of erythropoietin receptor. Nitric oxide induction of erythropoietin receptor promotes protection against hypoxia in neural cultures even in the absence of exogenous erythropoietin, although nitric oxide is toxic at high concentration. These data provide evidence of a role for nitric oxide in erythropoietin activity in brain and suggest links between nitric oxide production, erythropoietin receptor expression, and erythropoietin signaling in neuroprotection.